Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction

Abstract

A competitive polymerase chain reaction (PCR) method for quantification of white spot syndrome virus (WSSV) genome was developed. A pair of WSSV primers, designated WSSV341F/R, was selected to amplify a 341-bp DNA fragment from the WSSV genome. For a competitive internal standard, we constructed and cloned a 289-bp DNA fragment, the result of a 52-bp deletion from the 341-bp amplicon. In a competitive PCR reaction, we co-amplified the target WSSV DNA with known concentrations of the internal standard using WSSV341F/R primers. The amplicons from WSSV DNA and from internal standard DNA differed in size and could be distinguished after gel electrophoresis. The concentration of WSSV genomes was determined from its relation to the concentration of the internal standard. The log–log plot of the ratio of the amplicons (internal standard: WSSV) on the internal standard concentration was linear. Using this competitive PCR procedure, we quantified WSSV DNA in the samples of hemolymph and tissues of the cephalothorax of individual WSSV-infected shrimp. The number of WSSV genomes in both hemolymph and tissues corresponded to the severity of infection determined by histological evaluation. In addition, the changes in number of WSSV genomes in the hemolymph during the course of the infection were determined.